Non-viral vectors, although generally less toxic and immunogenic than the viral counterparts, are not without toxicity. The project is to design strategies to reduce the inflammation without sacrificing the gene transfer activity of the vector. In the last granting period, we have discovered that sequentially injecting cationic liposomes and plasmid DMA into the blood of mice significantly reduces the level of pro-inflammatory cytokines, such as TNFalpha. We were also able to mimic the activity by adding liposomes and DNA sequentially into freshly prepared mouse serum, suggesting the formation of new serum protein-containing complex which transfects cells but does not induce toxicity. The new complex also targets DNA mostly to the lung (the target organ), rather than the liver. Lipoplex added to the same serum also form complex with serum proteins, but the complex targets to the liver and secondarily to the lung. Preliminary proteomics data indicate that the lung-tropic complex contained unique serum proteins that were absent in the liver-tropic complex. One such protein was apolipoprotein A1 (apoA1). Thus, we hypothesize that these unique proteins, including apoA1, are responsible for the in vivo trafficking of DNA. In the aim 1 of the renewal application, we will continue to identify these proteins, prepare them using recombinant technology and test if any of these proteins will provide the desirable trafficking pattern in mouse. Since apoA1 is already identified as one of the proteins, we will characterize the complex containing this protein. A structure model for the complex which mimics HDL discoid is proposed and will be tested by biophysical methods. The interaction of the complex containing apoA1 with mouse lung endothelial cells will also be studied with an emphasis on the HDL receptor, i.e., SR-BI. In a separate effort, we have shown that dexamethasone and other anti-inflammatory drugs could be co-formulated in the lipoplex. Since the drug is co-delivered with the DNA into macrophages, their activity in producing the pro-inflammatory cytokines is significantly reduced. The gene transfer activity to the lung was not compromised. We named the drug-containing lipoplex as "safeplex", because it does not induce inflammatory toxicity. In aim 2, we will further improve the activity of the safeplex by synthesizing ethyl esters of several selected anti-inflammatory drugs that target to NF-kappa B, COX-2 and AP1. The hypothesis is that the ethyl ester, more hydrophobic than the drug itself, can be incorporated efficiently into the lipoplex and that it will be converted back to the drug once inside the macrophages and efficiently inhibit the inflammation mechanism. Lastly, we have discovered a few transcriptional represser binding sites in the standard plasmid DNA. Oligonucleotide decoys for these sites significantly enhance the transgene expression when codelivered with the plasmid DNA. Thus, the aim 3 will identify more represser sites and remove them from the plasmid vector by mutagenesis. The resulting plasmid should be more active in gene expression. The goal is to design safer and more efficacious non-viral vectors for gene therapy.